The neural circuitry of the dorsal horn of the spinal cord forms the basis for the mechanisms of pain and analgesia. Our lab has made significant inroads in understanding the neuronal connectivity which subserves these sensory phenomena through experiments involving multiple markers to identify interactions between neural elements. The anatomical features of calcitonin-gene-related peptide (CGRP), a novel peptide which may arise exclusively from primary afferent axons, was examined. Dense CGRP immunoreactivity was observed in the superficial dorsal horn and in lamina V. Ultrastructurally CGRP varicosities appeared either as central endings of glomeruli or dome-shaped. They contained mainly oval granular vesicles and formed asymmetrical synapses on dendrites and cell bodies. Mainly unmyelinated axons contained CGRP, suggesting an important role in pain transmission. Coexistence of CGRP and substance P (SP) in axons in the dorsal horn was examined using immunologically distinct multiple fluorescent markers. The density of coexistent axons was greatest in laminae I, IIa and V. Since dorsal root ganglia neurons are the only likely source of the coexistent axons, our data suggest that SP primary afferent axons, which are important in nociception, terminate in these laminae. A second series of experiments attempted to quantify norepinephrine, enkephalin, substance P and serotonin inputs to serotonin raphe neurons. Serotonin neurons in the caudal raphe nuclei received a differential number of contacts both with respect to the neurochemical they contained and the location of contacts. A third series of experiments identified the location of opioid dorsal horn neurons which responded to chronic inflammation of the limb. Neurons in laminae I, II and V increased their level of opioid peptide as assayed by immunocytochemistry. Opioid neurons in other laminae appeared to respond less to this noxious input.